Local immune responses at mucosal surfaces are thought to be important in combating infection to many bacteria, viruses and parasites, which gain access to the host through these surfaces. However, stimulation of local immune responses with purified non-replicating antigens is difficult. In fact, the healthy immune system appears tolerant to non-living antigens when they are presented at mucosal surfaces. This has seriously inhibited the development of new vaccines that can be delivered mucosally. To date, only a few proteins have been identified which activate strong local and systemic immune responses following mucosal presentation. Of these, the highly homologous enterotoxins, Cholera toxin (CT) from Vibrio cholerae and heat labile toxin from enterotoxigenic E. coli-(LT) have been studied extensively [1, 2]. In addition to the generation of strong anti-toxin responses, these proteins are able to activate immune responses to normally non-immunogenic co-administered antigen [3]. The adjuvant activity of these toxins has been studied extensively in animal models. Their high toxicity, however, makes them impractical for use in humans.
To circumvent the toxicity problem, site directed mutants of these toxins have been engineered which are no longer enzymatically active but which retain adjuvant activity [4-7]. It is hoped that these non-toxic derivatives will be suitable for inclusion in new vaccine formulations. A number of such mutant toxins have been constructed and characterised. This work has helped to establish a structure/function relationship between adjuvant activity of these proteins and the holotoxin formation [8]. However, little time and effort has been spent on the identification of alternative adjuvants.
Pneumolysin produced by Streptococcus pneumoniae is a pore forming protein produced by the majority of the disease causing serotypes. Vaccination with this protein has been shown to elicit some protective effects in animal models of infection.
Pneumolysin has at least two major activities important in its role in pathogenesis: the ability to form pores and the ability to activate the complement pathway. The functional regions for these activities have been located within the molecule and both activities have been shown to be important in the causation of disease [9]. The toxin is important in the pathogenesis of meningitis as it can cause damage to the ependymal cilia of the brain [10] and can induce apoptosis of brain cells [11]. The neurotoxity of pneumolysin has been shown to be due to alterations in calcium flux into cells and signalling via activation of the p38 MAP kinase [11, 12]. Pneumolysin has recently been shown to bind to Toll-like receptor 4 (TLR-4) [13]. This interaction with TLR-4 was essential for the protection of mice against invasive disease caused by the pneumococcus. Pneumolysin therefore plays a diverse and important role in the pathogenesis of pneumoccocal infections.
Listeriolysin O has previously been used to deliver heterologous antigen peptides to the MHC class I antigen presentation pathway in order to stimulate cytotoxic T cell responses against the heterologous antigen. In this system, nucleic acids encoding the listeriolysin O protein fused to the heterologous antigen (e.g. a tumour-specific antigen or viral antigen) were administered directly to the subject as DNA vaccines, or used to transform the intracellular bacteria Listeria monocytogenes which were themselves used as live vaccines. In either case, it is believed that the fusion protein is produced within the vaccinated subject's own cells (either by the cells themselves, or by the infecting bacteria), degraded in the usual manner, and presented to the immune system via MHC class I molecules. See [14] and references cited therein.